1

Supplementary Information

Oomycete Pathogens Encode RNA Silencing Suppressors

Yongli Qiao1,2, Lin Liu3,4, Qin Xiong5, Cristina Flores1, James Wong1,2, Jinxia Shi1, Xianbing

Wang1, Xigang Liu3, Qijun Xiang1, Shushu Jiang1, Fuchun Zhang4, Yuanchao Wang5, Howard S.

Judelson1,2, Xuemei Chen2,3,6, Wenbo Ma1,2,*

1. Department of Plant Pathology and Microbiology, University of California, Riverside, CA

92521

2. Center for Plant Cell Biology, University of California, Riverside, CA 92521

3. Department of Botany and Plant Sciences, University of California, Riverside, CA 92521

4. College of Life Science and Technology, Xinjiang University, Urumqi, China, 830046

5. Department of Plant Pathology, Nanjing Agriculture University, Nanjing, China, 210095

6. Howard Hughes Medical Institute, University of California, Riverside, CA 92521

* Corresponding author:

Wenbo Ma

Phone: 951-827-4349

Fax: 951-827-4294

Email: wenbo.ma@ucr.edu

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figures

Supplementary Figure 1. Expression of PSR1 and PSR2 genes upon Phytophthora sojae

infection of soybean roots. Transcript levels of PSR1 and PSR2 were determined by semi-

quantitative RT-PCR during a time course (0-24 hours post infection) using total RNAs extracted

from roots infected with P. sojae strain P6497. Soybean actin gene was used as an internal

control.

0 8 12 16 20 24 hpi

PSR1

PSR2

Actin

ladder

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Supplementary Figure 2. GFP siRNA accumulation in the leaves of N. benthamiana 16c plants

infiltrated with Agrobacterium tumefaciens carrying 35S-GFP and individual viral or

Phytophthora effectors. Leaves at a similar developmental stage and without Agro-infiltration

were used as the mock control. The numbers represent relative siRNA abundance. Data from one

representative experiment are shown in Fig. 1b and results from three additional independent

biological replicates are shown here. Avh36 is a P. sojae effector that may also possess RNA

silencing suppression activity but has not been focused in this paper because it did not show

consistent suppression of RNA silencing as the other two PSRs did.

GFP

1 0.2 0.1 0.4 0.4

U6

GFP siRNA

Repeat 1

1 0.6 0.2 0.7 0.5 U6

GFP siRNA

1 0.4 0.2 0.9 0.6 U6

GFP siRNA

Repeat 2

Repeat 3

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 3. Systemic silencing of GFP suppressed by PSR1 in N. benthamiana

16c plants. Leaves of N. benthamiana 16c plants were infiltrated with Agrobacterium

tumefaciens carrying 35S-GFP and one of the following constructs: empty vector (EV), 35S-

CMV2b (positive control), 35S-PSR1, 35S-PSR1M and 35S-PSR2. The table specified the

numbers of completely and partially silenced plants on the systemic tissues. For leaves

expressing PSR1 or CMV2b, both complete and partial suppression of GFP silencing (lower

panel) were observed. Systemic silencing for these partially silenced plants was restricted to the

regions on and around the veins of the leaves.

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 4. PSR1 contains a potential nuclear localization signal (NLS) and is

located in the nucleus in plant cells. (a) Schematic map showing motif architectures of PSR1. SP,

signal peptide region; RXLR, RxLR/dEER host targeting motif; and NLS, bipartite nuclear

localization signal. The mutant PSR1M was generated by replacing all 16 amino acid residues in

the NLS motif to alanines. (b) Subcellular localizations of the PSR1-YFP and PSR1M-YFP

fusion proteins were determined in N. benthamiana leaves after Agrobacterium-mediated

transient expression. Yellow fluorescence was detected by confocal microscopy from infiltrated

tissues 48 hours post Agro-infiltration. Nuclear region in the red box was enlarged to better

visualize the lack of PSR1M proteins in the nucleoplasm.

PSR1-YFP

PSR1M-YFP

PSR1M-YFP (Enlarged)

Fluorescence Bright-field Merged

30 μm

30 μm 30 μm 30 μm

5 μm

30 μm 30 μm

5 μm 5 μm

a

b

SP RXLR NLS

PSR1 RKMLGDETYRLKKFGK AAAAAAAAAAAAAAAA

52 37

PSR1M

PSR1 106

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 5. PSR1 and PSR2 do not possess small RNA binding activity in vitro.

Electrophoretic mobility shift assays were performed using purified GST-tagged recombinant

proteins and 32P-labelled synthetic small RNA oligos that were 21 nt in length. The viral RNA

silencing suppressor P19 that is known to bind to ds-sRNA was used as a positive control.

ss-sRNA ds-sRNA

- GST P19 PSR1 PSR2

Free ss-sRNA

Free ds-sRNA

P19 ds-sRNA Complex

- GST P19 PSR1 PSR2

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 6. Semi-quantitative RT-PCR showing the expression of PSR1 and

PSR2 in transgenic Arabidopsis plants. Three independent transgenic lines (as indicated by the

different numbers) harboring 35S-PSR1-YFP or 35S-PSR2-Flag were assayed for transgene

expression. Col-0, wild-type. Inflorescence tissues were used to confirm the expression of PSR1-

YFP and PSR2-Flag in the transgenic lines by RT-PCR with UBQ5 as an internal control. The

three lines with varying expression levels for each construct were further investigated for small

RNA levels (Fig. S7, S9).

Col-0 PSR1-6 PSR1-19 PSR1-22

Col-0 PSR2-5 PSR2-10 PSR2-18

PSR1

UBQ5

PSR2

UBQ5

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 7. Reduced accumulation of small RNAs in Arabidopsis transgenic

lines expressing PSR1. a, PSR1-expressing plants are much smaller with other morphological

phenotypes. Pictures were taken on 20-day-old Arabidopsis plants grown in soil. Col-0, wild-

type; PSR1-6, PSR1-19, PSR1-22, three independent 35S-PSR1-YFP Arabidopsis transgenic

lines. b, Levels of representative miRNA, trans-acting siRNA (ta-siRNA) and heterochromatic

siRNA (hc-siRNA) species in wild-type (Col-0) and the transgenic lines were determined by

northern blots using total RNAs extracted from inflorescences. U6 served as an internal control

for each blot. The numbers below the gel images represent relative small RNA abundance.

Col-0

PSR1-6

PSR1-19

PSR1-22

1 0.8 0.5 0.5

1 0.7 0.6 0.6

b

U6

Cluster4

SimpleHAT2

U6

U6

AtSN1

1 0.6 0.5 0.4

1 0.8 0.5 0.7

1 0.7 0.2 0.5

ASRP255

U6

U6

ASRP1151

miR164

U6

miR159

miR167

U6

miR171

miR390

1 0.4 0.3 0.3

1 0.4 0.3 0.3

1 0.5 0.3 0.3

1 0.4 0.2 0.3

1 0.4 0.3 0.3

miR166

U6 1 0.7 0.4 0.5

miRNAs

Col-0 PSR1-6 PSR1-19 PSR1-22

ta-siRNAs

hc-siRNAs

a

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Supplementary Figure 8. Transcript levels of miRNA or ta-siRNA target genes were

determined by quantitative RT-PCR in wild-type (Col-0) and representative transgenic

Arabidopsis lines expressing PSR1 or PSR2. AGO1, CUC2, PHV, SCL6, and SPL10 are targets

of miR168, miR164, miR165/166, miR171, and miR156/157, respectively. At4g29770,

At1g12775, and ARF3 are targets of ta-siRNAs from TAS1, TAS2, and TAS3, respectively. Total

RNAs extracted from inflorescences were used for this analysis.

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 9. PSR2 does not affect the accumulation of miRNAs or

heterochromatic siRNAs (hc-siRNAs) in Arabidopsis. (a) Expression of PSR2 does not

significantly change the morphological phenotype in Arabidopsis. Pictures were taken on 20-

day-old Arabidopsis plants grown in soil. Col-0, wild-type; PSR2-5, PSR2-10, PSR2-18, three

independent 35S-PSR2-Flag Arabidopsis transgenic lines. (b) Levels of representative miRNAs

and heterochromatic siRNAs (hc-siRNAs) in wild-type and 35S-PSR2-Flag transgenic

Arabidopsis lines were determined by northern blotting. The numbers below the images

represent relative abundance of the small RNAs compared to wild-type (Col-0). U6 served as an

internal loading control.

Col-0

PSR2-5

PSR2-10

PSR2-18

a b Col-0 PSR2-5 PSR2-10 PSR2-18

miRNAs

hc-siRNAs

U6

miR166

miR167

U6

miR171

miR390

U6

Cluster4

SimpleHAT2

AtSN1

U6

U6

1 1.2 1.1 0.9

1 1.0 1.1 0.9

1 1.2 1.3 1.0

1 1.2 1.4 1.3

1 1.0 0.8 1.0

1 0.7 0.8 1.1

1 0.7 0.8 1.3

U6

miR164

miR159 1 1.0 1.1 1.0

1 0.9 1.1 1.1

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 10. AGO1-miR173 slicer activity assay in wild-type (Col-0) and two

independent PSR2-expressing Arabidopsis lines. A portion of the TAS1c locus containing the

miR173 target site was amplified by PCR with one of the primers containing a T7 promoter

sequence. The PCR fragment was used as a template for in vitro transcription by T7 polymerase

to generate a 192 nt TAS1c transcript, which was incubated with AGO1 immunoprecipitates from

Col-0 or two PSR2-expressing lines. AGO1/miR173-mediated cleavage of the 192 nt transcript

results in a 170 nt fragment and a 22 nt fragment, the latter beyond the range of detection. This

assay showed that PSR2 did not affect the slicer activity of AGO1/miR173 in Arabidopsis.

TAS1C Col-0 PSR2-5 PSR2-10

192 nt

~170 nt

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Supplementary Figure 11. PSR1 promotes the infection of N. benthamiana by potato virus

X (PVX). N. benthamiana plants were infiltrated with Agrobacterium tumefaciens carrying

35S-PVX, 35S-PVX-PSR1, PVX-PSR1M or 35S-PVX-ΔPSR1. The accumulation of PVX

genomic and subgenomic RNAs was analyzed at four days post inoculation. This experiment

was repeated twice with similar results. Data from one representative experiment are shown

in Fig. 3b; additional data from the other replicate are shown here.

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 12. PSR2 promotes PVX infection of N. benthamiana. (a) Disease

symptoms of N. benthamiana plants infiltrated with Agrobacterium tumefaciens strains carrying

35S-PVX, 35S-PVX-PSR2 or 35S-PVX-ΔPSR2. The plants infected with PVX-PSR2 exhibited

more severe mosaic symptoms compared to those expressing wild-type 35S-PVX or 35S-PVX-

ΔPSR2. The photograph was taken at 21 days post-infiltration (dpi). (b) Northern blot analysis

showing the accumulation of viral genomic (gRNA) and subgenomic mRNAs at 21 dpi. This

experiment was repeated twice with similar results.

a b

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 13. Over-expression of Phytophthora and viral RNA silencing

suppressors in N. benthamiana increases plant susceptibility to the infection of Phytophthora

infestans isolate 1306. Data from two independent biological replicates are presented showing

the lesion sizes and numbers of sporangia from N. benthamiana leaves expressing PSRs and

VSRs. In each experiment, leaves from 6-10 plants were used for each treatment. Error bars

represent standard errors and different letters indicate values with significant differences.

EVPS

R1PS

R1M

PSR2

CMV2

bP1

9HC

-Pro

Lesi

on D

iam

eter

(mm

)

0

5

10

15

20

25

30

EVPS

R1PS

R1M

PSR2

CMV2

bP1

9HC

-Pro

Spor

angi

a pe

r lea

f (x1

000)

0

10

20

60

70

80b

a

c

c c d

a a

b

d d

c

a

c a a

EVPS

R1PS

R1M

PSR2

CMV2

bP1

9HC

-Pro

Lesi

on D

iam

eter

(mm

)

05

101520253035

EVPS

R1PS

R1M

PSR2

CMV2

bP1

9HC

-Pro

Spor

angi

a pe

r lea

f (x1

000)

0

10

20

60

70

80

90b

a

d

c

d

a ac

b

d

d

c ac a

a

Repeat 1

Repeat 2

Nature Genetics: doi: 10.1038/ng.2525

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Supplementary Figure 14. Expression of the PSRs and VSRs in N. benthamiana leaves did

not elicit visible lesions or cell death symptoms without the subsequent infection of P.

infestans. The effector proteins were expressed in N. benthamiana by Agro-infiltration. 24

hours after the plants were Agro-infiltrated, the leaves were detached and incubated in a growth

chamber at 18°C. The photograph was taken seven days after Agro-infiltration.

PSR1 PSR2 CMV2b P19 HC-Pro EV PSR1M

Nature Genetics: doi: 10.1038/ng.2525

Supplementary Table 1. Bacterial strains and plasmids used in this study Strains or Plasmids Description Source/ reference Escherichia coli DH5α F- Φ80dlacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1, hsdR17(rk-, mk+) phoA supE44 λ- thi-1

gyrA96 relA1 Invitrogen

Escherichia coli BL21(DE3) F– ompT gal dcm lon hsdSB(rB- mB -) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5] Invitrogen Agrobacterium tumefaciens GV3101 (pMP90)

RifR, GentR 26

Agrobacterium tumefaciens C58C1 (pCH32)

RifR, TetR 26

Phytophthora infestans isolate 1306 A1 mating type Phytophthora sojae strain P6497 Race 2 strain with full genome sequenced 3, 27, Michael Coffey pCAMBIA1300::CMV2b pCAMBIA1300 carrying CMV2b, KanR 30, Shou-Wei Ding pCAMBIA1300::P19 pCAMBIA1300 carrying P19, KanR Shou-Wei Ding pCAMBIA1300::HC-Pro pCAMBIA1300 carrying HC-Pro, KanR Shou-Wei Ding pEG100 pEarleyGate100, a Gateway binary vector with cauliflower mosaic virus 35S promoter, KanR 28 pEG100::PSR1 pEG100 carrying PSR1, KanR This study pEG100::PSR1-YFP pEG100 carrying PSR1-YFP, KanR This study pEG100::PSR1M pEG100 carrying PSR1M (lacking the putative NLS motif), KanR This study pEG100::PSR2 pEG100 carrying PSR2, KanR This study pEG100::PSR2-Flag pEG100 carrying PSR2-Flag, KanR This study pEG101 pEarleyGate101, a Gateway binary vector with cauliflower mosaic virus 35S promoter and yfp, KanR 28 pEG101::PSR1 pEG101 carrying PSR1 tagged with YFP at the C-terminus, KanR This study pEG101::PSR1M pEG101 carrying PSR1M tagged with YFP at the C-terminus, KanR This study pGR106 a binary vectors carrying the Potato virus X genome, KanR 37, Sophien Kamoun pGR106::PSR1 pGR106 carrying PSR1, KanR This study pGR106::PSR1M pGR106 carrying PSR1M (lacking the putative NLS motif), KanR This study pGR106::ΔPSR1 pGR106 carrying ΔPSR1 (stop codon at the 3 aa position of the ORF), KanR This study pGR106::PSR2 pGR106 carrying PSR2, KanR This study pGR106::ΔPSR2 pGR106 carrying ΔPSR2 (stop codon at the 8 aa position of the ORF), KanR This study pGEX4T-2 E. coli expression vector with C-terminal GST tag, AmpR Amersham pGEX4T-2::PSR1 pGEX4T-2 carrying PSR1 that expresses GST-PSR1, AmpR This study

pGEX4T-2::PSR2 pGEX4T-2 carrying PSR2 that expresses GST-PSR2, AmpR This study pGEX4T-2::P19 pGEX4T-2 carrying P19 that expresses GST-P19, AmpR This study pTH209 Phytophthora transformation vector used as a helper vector for dsRNA-induced silencing of PSR2,

G418R, KanR 40

Nature Genetics: doi: 10.1038/ng.2525

Supplementary Table S2. Phytophthora sojae PsAvh genes screened for RNA silencing suppression activity in this study

Index Effector ID VMD ID Forward sequence Reverse sequence1* Avr1b AAM20936 2* PsAvh_5 158993 3* PsAvh_6 158994 4 PsAvh_8 158999 CTACGTCGACATGTTGTCAGCGGGCCCCGGCAAGG TCTACGAATTCGACTATGGGTACTGTCTAGTGTTCC5 PsAvh_16 159004 TCTACGTCGACATGGCCCTCCCTTCGGCCACGGCAT TCTACGAATTCGATCACATGCCCATCTTCTTTGCTT6 PsAvh_18 159006 CTACGAATTCGCATGACTAAACCGTCGACGGAGGC CTACGATATCTTCATTGTTCTAGCCACGCCT7 PsAvh_23 159011 TCTACGTCGACATGCTCGCCACCGCCGAGGCCCCCA TCTACGAATTCGATCATGCATTGTCGGAAAGTTTGA8 PsAvh_29 159017 TCTACGAATTCGCATGGCCGTCGCGGGTGCCAGCGA TCTACGATATCTTCAATGGAAATTATTGATGAGCCA9 PsAvh_35 159022 CTACGAATTCGCATGGCCGCGGGTGGTACTGCCGT CTACGATATCTCTAATGGTTGGTGAACCAGTA10 PsAvh_36 159023 CTACGAATTCGCATGCTCTCGACCACCACGGACTC CTACGATATCTTCAAGTGAGAGTCCTTCCTTCG11 PsAvh_38 159025 TCTACGAATTCGCATGGTTCAGGTCTCTCCAGCTGC TCTACCTCGAGTGCTATGTCCGTGTCGGATGTGCCT12 PsAvh_39 159026 CTACGTCGACATGGCTCAACCCACGCCAGTGAA CTACGAATTCGATTAGTCGATCTTCACCTTTCC13 PsAvh_42 159029 CTACGTCGACATGTTGAGTATCACCAATTCCGA CTACGAATTCGATCACTTTTTTGGGATCGACG14 PsAvh_52 159037 TCTACGTCGACATGCTCACGTTGACCAAGGATTCCA TCTACGAATTCGATCAGTTGGCCGCCTTATAAACCT15 PsAvh_63 159042 CTACGTCGACATGCAATCCTCCGCCACCGCCAC CTACGAATTCGATTACGTAGTCCTCTGGTGCA16 PsAvh_66 159045 CTACGTCGACATGGTTCCTGCTTCCGCAGACAT CTACGAATTCGATTAGGGGTATCTCAGGGGCC17 PsAvh_67 159046 CTACGAATTCGCATGCTCCCCGCAGACCAGGCGGC CTACGATATCTTTACAAAATCTCGCCAGTCTG18* PsAvh_73 159050 19* PsAvh_92(Avr3a) 159064 20 PsAvh_94 159065 TCTACGTCGACATGGCCAACACCAAGGTGGAGTCGT TCTACGAATTCGACTAAGCGGTGTCCTCCTCCTCCT21 PsAvh_105 159073 CTACGTCGACATGGCCTCCGATCTGACCACGACG CTACGAATTCGATTAAATCTTAAACTTGCCGAC22 PsAvh_109 159076 TCTACGAATTCGCATGCTTCAGGTCCTCCCCAATTC TCTACGATATCTTTAATCAGCGGTTTGTCGCCTGTA23 PsAvh_115 159081 TCTACGTCGACATGGTCCCAGACTCGAAGGTCTCAA TCTACGAATTCGACTAGGCGGCATTCTTGCGAGCAT24 PsAvh_122 159085 CTACGTCGACATGTTGGTCCTTGGGGAAGGTGT CTACGAATTCGATTAGACTGCCTCCATGACGC25 PsAvh_127 159090 CTACGTCGACATGGCGGGGCTCGCTGATGCATC CTACGAATTCGATTACTTTTTGCCAGAGTTGG26 PsAvh_137 159094 TCTACGTCGACATGGCCGTGGCCGATCCCAAGAGCT TCTACGAATTCGATCACGGGTAAATGTATCCCTCAA27 PsAvh_138 159095 CTACGAATTCGCATGCTTCAACCGGGACAGATCGC CTACGATATCTTTACTGCTTACTACACCAATA28 PsAvh_139 159096 CTACGTCGACATGGCGTCTGCTGCCACAGAATC CTACGAATTCGATTAGGGATTGAAAGAAAATA

29 PsAvh_140 159097 TCTACGGATCCGGATGGAATTCCCTTCGGTGGCTTC TCTACGATATCTCTACTGTTTGCTGCTGGTCAGCTC30 PsAvh_145 159101 CTACGAATTCGCATGACACCAGCATCTCTACTAAC CTACGATATCTCTACTTCTTGAGACGTTTGAAGGC31 PsAvh_146 159102 CTACGAATTCGCATGACACATGCTCCTCCTAACGT CTACGATATCTTTACCCCCACCTGACTTTGAACTT32 PsAvh_147 159103 CTACGTCGACATGTCGGTGACAGATAACTCAATCG CTACGAATTCGATTAGGTCGCCCTGTTCGTGCGCA33 PsAvh_148 159104 CTACGTCGACATGGAATCTGTAGGCCCCATCAC CTACGAATTCGATCACCGCCTAGAAGGATATTTCA34 PsAvh_151 159107 CTACGAATTCGCATGGCACCCGAGTCTGCCACCGC CTACGATATCTTCACTGAATCTTCGTACCTTTGAG35 PsAvh_156 159112 CTACGTCGACATGGCCTCGGCGACTTCGACGCT CTACGAATTCGACTACAGCAGATACAGCTTGTATG36 PsAvh_162 159118 CTACGTCGACATGAGCACCGACTCCAAGATCGT CTACGAATTCGATTATTTTGCCCTTACGTTCATGG37 PsAvh_163 159119 CTACGTCGACATGGCTACCGTATCAGCACTCCAGC CTACGAATTCGACTAAGAGCGCCGAGGGGTCACAT38 PsAvh_170 159126 CTACGTCGACATGAGGACTGTGGTGACGCCTAGTT CTACGAATTCGACTAGGCACTGTATGCAGCTCGGA39* PsAvh_171(Avr4/ 159127 40 PsAvh_172 159128 TCTACGAATTCGCATGGAGGTCGACTCGAAGACGGC TCTACGATATCTCTATTCCCCCCGTAATTTGTAAAA41 PsAvh_180 159136 TCTACGTCGACATGGAGACCCAGCTTTCGCAGACGA TCTACGAATTCGACTAAGCGATGTTCGTCTGCTTCT42* PsAvh_181 159137 43 PsAvh_182 159138 CTACGTCGACATGCTACCGGCCTCCGACCAACT CTACGAATTCGACTAGTGGGGGAATTGTTGCGAGT44 PsAvh_183 159139 CTACGTCGACATGGAGTCTGCGTCCACAGATACTA CTACGAATTCGATTACGGGTAGCTTCTATGGATCG45 PsAvh_189 159145 CTACGTCGACATGACCGCGACCGTCACCGACTCGA CTACGAATTCGATCAGTAGTCGCAACCCGAACTTA

46 PsAvh_190 159146 CTACGAATTCGCATGACTACCGTCTCGGACAAGTC CTACGATATCTCTACGGGGCAAAGATGTGGATGGG

47 PsAvh_194 159150 CTACGTCGACATGCTTTCAGCGGCCACGGAGATCGA CTACGAATTCGATTATCCTCGTGGGGCACCACCTG48 PsAvh_196 159152 TCTACGAATTCGCATGTCGGTTAGCAACCTCCAAAC TCTACCTCGAGTGTTAGCTGAGCTTGGACGTCCACC49 PsAvh_205 159161 CTACGTCGACATGGACGTCGTCTCGGCCTCCATCG CTACGAATTCGATTAAACGACGAGCTTGCTAGCCC50 PsAvh_231 159187 TCTACGAATTCGCATGGCCGTGCATACCCAAGAAGG TCTACCTCGAGTGCTAGCGAATCGATGTGCTTCTGG51* PsAvh_238 159194 52 PsAvh_240 159196 TCTACGTCGACATGGACGCCGGCCGAGTTCTCGAGA TCTACGAATTCGACTAGTTTGCGGGTTGGTTCCGGA53 PsAvh_244 159200 CTACGTCGACATGGCCAGGAACCGTGCACCCGGGA CTACGAATTCGATCACGCGACTTTCGCCACAGCTT54 PsAvh_254 159210 CTACGAATTCGCATGGAGAAGGCCGTCGACGTGGA CTACGATATCTTCAGTTGGCTCGTGGGCTGCCCAT55 PsAvh_256 159212 TCTACGTCGACATGGAGTCCGCAAACGGCGTTTCCC TCTACGAATTCGATTAAGTGGTCTTTTTAGCAGCGG56 PsAvh_260 159216 TCTACGTCGACATGGCCACGTGCATCAGTGCCTCGA TCTACGAATTCGATTAGTCCTTGAGCATGGCGGTGA57 PsAvh_263 159219 TCTACGTCGACATGGATGACACGCACCGAAGCCT TCTACGAATTCGACTAATGGATATAACTGTTCTTGA58* PsAvh_275(Avr1a 159231 59* PsAvh_331(Avr1k 159287

* effector constructs provided by Dr.Brett Tyler

Primers used in this study for gene cloning

Nature Genetics: doi: 10.1038/ng.2525

Supplementary Table 3. Primers used in this study.

Primer name Primer sequence (5’ to 3’) pGR-PSR1ClaI-F CTACATCGATATGACTAAACCGTCGACGGAGGC pGR-ΔPSR1ClaI-F CTACATCGATATGACTAAACCGTAGACGGAGGC pGR-PSR1NotI-R CTACGCGGCCGCTTTGTTCTAGCCACGCCTTGT pGR-PSR2AscI-F CTACGGCGCGCCATGACACATGCTCCTCCTAACGTTAAG pGR-ΔPSR2AscI-F CTACGGCGCGCCATGACACATGCTCCTCCTAACGTTTAG

PVX assay

pGR-PSR2NotI-R CTACGCGGCCGCACCCCCACCTGACTTTGAACTT PVXCP-F GTCAACTACCTCAACTACCAC PVXCP-R TATGTAGACGTAGTTATGGTGG GFP-F GAAGGTGATGCAACATACGG

Northern blotting

GFP-R TCCATGCCATGTGTAATCCC PSR1-RT-F ACTAAACCGTCGACGGAGGCGACTG PSR1-RT-R TCATTGTTCTAGCCACGCCTTGTAC PSR2-RT-F ACGAGGTTCTGTCGGGTATG PSR2-RT-R GTCAAGCGATAGCAACGTGA AtUBQ5-F GGTGCTAAGAAGAGGAAGAAT AtUBQ5-R CTCCTTCTTTCTGGTAAACGT GmActin-F GTTCTCTCCTTGTATGCAAGTG

RT-PCR

GmActin-R CCAGACTCATCATATTCACCTTTAG PSR2-QPCR-F TGTTCGCGGCAAAGAAGGAC PSR2-QPCR-R CCTGACTTTGAACTTGGCGG PsojaeActin-F ACTGCACCTTCCAGACCATC

Q-RT-PCR

PsojaeActin-R CCACCACCTTGATCTTCATG AGO1-F TGGACCACCGCAGAGACAAT AGO1-R CATCATACGCTGGAAGACGACT CUC2-F TTTTCCTCGTTTCGTTTCTA CUC2-R TCCAAATACAGTCAAGTCCA PHV-F CAAGGCTACAGGAACTGC PHV-R TGAGGATTTCAGCGACCT SCL6-F ACTCAAGACAACCTCAAGCA SCL6-R GATAGATGCTTCACGAAACG SPL10-F TGAGACAAAGCCTACACAGATGGA SPL10-R GATGATGCAACCCGACTTTTTTATG At4G27990-F CCGTCAGGTAATGAAACAC At4G27990-R TGGGATACAGAAGTCAACAA At1G12775-F GCTTTTTCTACTATGGGGAAG At1G12775-R ATGAGAGTGGGTTTATGTCC ARF3-F GGTGGCCTGGTTCAAAATGGAG

sRNA target real-time RT-PCR

ARF3-R CGGAAGAGGGTGATGATGATAC pre-miR164bF GATGGAGAAGCAGGGCACGT pre-miR164bR GTGAAGATGGGCACATGAAG pre-miR166aF AGATATATATTCAGAAACCCTAG

pre-miRNA northern blotting

pre-miR166aR GGTTCATTCACTGGATCTGAAAC PSR2-F GTAATACGACTCACTATAGGGACGAAGAGCGGGGAATCAACT PSR2

silencing PSR2-R GTAATACGACTCACTATAGGGGTCCCTGTCTTGTCAAGTCGT

Nature Genetics: doi: 10.1038/ng.2525

Primer name Primer sequence (5’ to 3’) miR159 TAGAGCTCCCTTCAATCCAAA miR167 TAGATCATGTTGGCAGTTTCA miR319 GGGAGCTCCCTTCAGTCCAA miR163 ATCGAAGTTCCAAGTCCTCTTCAA miR164 TGCACGTGCCCTGCTTCTCCA miR171 CGTGATATTGGCACGGCTCAA miR172 ATGCAGCATCATCAAGATTCT ASRP255 TACGCTATGTTGGACTTAGAA ASRP1151 AAGTATCATCATTCGCTTGGA ASR5D8 AAAGGCCTTACAAGGTCAAGA miR173 GTGATTTCTCTCTGTAAGCGA miR393 GATCAATGCGATCCCTTTGGA miR166 GGGGAATGAACGCTGTTCGCT miR390 GGCGCTATCCCTCCTGAGCTT miR168 TTCCCGACCTGCACCAAGCGA siR1003 ATGCCAAGTTTGGCCTCACCGTC AtSN1 ACCAACGTGTTGTTGGCCCAGTGGTAAATCTCTCAGAT SimpleHAT2 TGGGTTACCCATTTTGACACCCCTA Cluster4 AAGATCAAACATCAGCAGCGTCAGAGGCTT

miRNA Northern blotting

U6 AGGGGCCATGCTAATCTTCTC pri-miR159F GGAGCTCTACTTCCATCGTCA pri-miR159R CCACGTTCTCATCAAAACTTT pri-miR163F GCATAGGTCTTGATTGGTGGA pri-miR163R CGTTGTCGTTGAAGAGGTTG pri-miR164F CCATTGACGATTGCATCCTCG pri-miR164R TTGATGGAGAAGCAGGGCAC pri-miR166aF CACCACTCACTTATCTTCTTC pri-miR166aR CAGTCGAAATTATAGAATCTAGGGT pri-miR167F GAAGCTGCCAGCATGATCTA pri-miR167R GGGTTTATAGAAGGGTGCGA pri-miR171F CCGCGCCAATATCTCAGTA pri-miR171R TGTCTCCATTTCAACACACACA pri-miR172F TAGGGTTAGCATGTTGATGAC pri-miR172R CCTCAAGTTATCATATCGGAG pri-miR173F CTTCTTCTCACAAATAAACCCA

pri-miR173R AAGATCTCTAACATTAAATCAT pri-miR319F AGAGGTTAGCATGTTGATGAC

pri-miRNA Q-RT-PCR

pri-miR319R CCTCAAGTTATCATATCGGAG TAS1CF TAATACGACTCACTATAGGGTTAGTTTGAGATTGCGTTTGTC AGO1 slicer

assay TAS1CR CTAAGATCCACCGATAAATGGTC si21-1 CGUACGCGGAAUACUUCGAUU

si21-2 UCGAAGUAUUCCGCGUACGUU

P19-BamHI-F CTACGGATCCATGGAACGAGCTATACAAGG

P19-XhoI-R CTACCTCGAGTTTACTCGCTTTCTTTCTTGA

PSR1-BamHI-F CTACGGATCCATGACTAAACCGTCGACGGAGGC

PSR1-EcoRI-R CTACGAATTCGTCATTGTTCTAGCCACGCCT PSR2-BamHI-F CTACGGATCCATGACACATGCTCCTCCTAACGTTAAG

EMSA

PSR2-EcoRI-R CTACGAATTCGTTACCCCCACCTGACTTTGA

Nature Genetics: doi: 10.1038/ng.2525